Project Summary Ischemia reperfusion injury (IRI) is a major complication after liver surgery. In liver transplantation, it is a major risk factor for both acute and chronic graft rejection and restrains the use of marginal donors. Liver inflammatory immune response drives the pathogenesis of IRI. Despite the progress in studies of cellular and molecular mechanisms of the response, questions regarding to the potential distinctive roles of tissue resident vs. infiltration macrophages (iM?s), and the resolution of liver IRI remain to be defined. Glycogen synthase kinase 3? (Gsk3?) is a unique signaling kinase that it differentially regulates pro- and anti-inflammatory gene programs in M?s upon innate stimulations. It has been shown in a murine liver partial warm ischemia model that both Gsk3 inhibitors in WT hosts and myeloid Gsk3? deficiency protect livers from IRI via an AMPK- IL-10-dependent mechanism. The clinical relevance of AMPK in liver ischemia has been documented recently in hepatic tumor resection. Preliminary experiments of this proposal have found that Gsk3? also regulates the resolution of liver IRI. In fact, Gsk3? regulates liver inflammatory immune activation and resolution by targeting distinctive populations of liver M?s. KCs depletion diminished the liver protective phenotype in myeloid Gsk3? KO mice against IR. However, the recovery of liver IRI remain accelerated in these KC-depleted KO hosts, indicating that Gsk3? regulates iM?s in resolving tissue inflammation. Current proposal will dissect mechanisms of Gsk3? regulation of these two types of M?s at different stages of liver IRI. IR-induced liver inflammatory immune activation is characterized by infiltration and activation of M?s from peripheral (iM?s), as well as necroptotic depletion and pro-inflammatory conversion of tissue resident KCs. In liver inflammation resolution, M?s repair hepatocellular damage and clear infiltrated neutrophils via Tyro3-Axl-MerTK (TAM) receptor tyrosine kinase (RTK)-mediated efferocytosis. Macrophage necroptosis and efferocytosis potently regulate their activation and differentiation. Gsk3? promotes stress-induced cell death and regulates TAM expressions in M?s. The proposal will test the hypothesis that Gsk3? regulates KC necroptosis via AMPK and pro-inflammatory immune activation via Mer RTK; and Gsk3? inhibition/inactivation facilitates iM? conversion from pro-inflammatory to reparative type by enhancing Axl-mediated efferocytosis and immune regulatory signaling. Two specific aims are designed to address how Gsk3? regulates KC inflammatory activation, and iM? reprogramming/functions, in the activation and resolution stages of liver IRI, respectively. Results of these experiments shall offer fresh new insight into immune regulatory functions of Gsk3? in M?s in both the activation and resolution of liver inflammation and injuries and provide rationales for novel therapeutic strategies to restore liver homeostasis in patients.